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Distinctive Gene and Protein Characteristics of Extremely Piezophilic Colwellia Logan M. Peoples1, Than S. Kyaw1, Juan A. Ugalde

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Distinctive Gene and Protein Characteristics of Extremely Piezophilic Colwellia Logan M. Peoples1, Than S. Kyaw1, Juan A. Ugalde Distinctive Gene and Protein Characteristics of Extremely Piezophilic Colwellia Logan M. Peoples1, Than S. Kyaw1, Juan A. Ugalde2, Kelli K. Mullane1, Roger A. Chastain1, A. Aristides Yayanos1, Masataka Kusube3, Barbara A. Methé4, Douglas H. Bartlett1* Supplementary Information, bioRxiv Supplementary Figure 1. Growth curves of strains of Colwellia psychrerythraea as a function of pressure and temperature. A, C. psychrerythraea 34H; B, C. psychrerythraea ND2E; C. psychrerythraea GAB14E. A 4 °C 16 °C B 4 °C 16 °C C 4 °C 16 °C 0.30 0.30 0.30 0.25 0.25 0.25 Pressure 0.1 MPa 0.20 0.20 0.20 20 MPa 0.15 0.15 0.15 40 MPa 60 MPa 0.10 0.10 0.10 80 MPa Optical density (OD600) 0.05 0.05 0.05 0.00 0.00 0.00 0 5 10 0 5 10 0 5 10 0 5 10 0 5 10 0 5 10 Time (days) Tree scale: 0.01 Pseudomonas aeruginosa (X06684) Shewanella benthica (AB238792) Pseudoalteromonas atlantica (NR 026218) Thalassotalea atypica (NR 159308) 0.96 Thalassotalea crassostreae (NR 157655) Thalassotalea sediminis (NR 149198) 0.979 Thalassotalea piscium (NR 125636) 0.994 1 Thalassomonas agariperforans (HM237288) 0.974 Thalassomonas ganghwensis (AY194066) Thalassomonas loyana (AY643537) 0.997 Thalassomonas agarivorans (DQ212914) Thalassotalea euphylliae (NR 153727) 0.926 Thalassomonas haliotis (AB369381) 0.958 Thalassomonas viridans (AJ294748) 0.918 Thalassomonas actiniarum (AB369380) Colwellia aquaemaris (JQ948043) 0.998 Colwellia meonggei (KF193862) Colwellia sp. JAM-GA24 (AB526347) Colwellia aestuarii (DQ055844) 0.965 Colwellia sp. STAB104 (JF825438) 0.89 0.857Colwellia sp. P12 (EF627997) 0.797 0.783 Colwellia sp. P5 (EF627991) Colwellia chukchiensis (FJ889599) 0.993 Colwellia polaris (DQ007434) 0.884 Colwellia sp. Arc7-D (2791048221) 0.88 Colwellia sediminilitoris (NR 151990) 0.982 Colwellia mytili RA2-7 (NR 156046) Colwellia beringensis (NR 159245) 0.986Colwellia sp. BSw20736 (GU166126) 0.925 Colwellia sp. ANT8279 (AY167268) 0.999 Colwellia sp. E4-4 (FN377730) Colwellia asteriadis (EU599214) Colwellia demingiae (U85845) Colwellia psychrerythraea 34H (640698408) 0.981 0.978 Colwellia psychrerythraea GAB14E (2619430172) 0.861 Colwellia psychrerythraea ND2E (2619423572) 0.964 Colwellia echini (NR 159353) 0.991 0.949 Colwellia arctica (NR 136835) Colwellia hornerae PAMC 20917 (2719491234) Colwellia piezophila (2521854739) 0.97 Colwellia sp. TT2012 (2587690801) 0.941 1 Colwellia sp. KT27 (DQ027052) 0.993 Colwellia marinimaniae MTCD1 (2585890133) 1 Colwellia marinimaniae MT41 (DQ027051) Colwellia maris (AB002630) 0.852Colwellia sp. KJF9-8 (JQ800146) 0.936 Colwellia psychrotropica (U85846) 0.991 Colwellia rossensis (U14581) Supplementary Figure 2. Ribosomal 16S RNA gene tree of members of the Colwellia. Strains in bold were compared in this study; blue are piezophilic, black are piezosensitive. 42 A 42 B C 54.040 6.0 53.5 5.5 Type 40 AtmosphericOther growth ObligatePiezophile piezophile 53.0 5.0 Genome %GC 38 38 Genome %GC 52.5 (Mb) Genome size 4.5 16S rRNA gene %GC 36 52.0 4.0 36 Supplementary Figure 3. Strains of Colwellia as a function of (A) their genome % GC, (B) full length 16S rRNA gene % GC, and (C) genome size. A Colwellia Psychromonas Shewanella 55 loihica algae amazon carassii chilikensis 50 mangrovii bice decolorationis baltica KT99 colwelliana oneidensis zor a Piezophile 45 waksmaniidenitrificans violacea GC (%) piezotoleransputrefaciens a Piezosensitive aquimarina psychrophila fidelis chukchiensis ossibalaenae mb_3u_54 aestuarii frigidimarina 40 TT2012 japonica beringensis mytili MT41 ingrahamii CNPT3 MTCD1 ND2E 34H urea hadalis piezophila prt−sc03 echini 12g3 hornerae arctica arc7 polaris GAB14E 35 agarivorans −40 −20 0 −40 −20 0 −40 −20 0 ← Increasing acidic pI bias (%) B 1 2 3 4 55 amazon algae loihica carassii chilikensis 50 mangrovii decolorationis baltica colwelliana denitrificans KT99 zor oneidensis violacea 45 waksmanii a Piezophile putrefaciens piezotolerans psychrophila GC (%) a Piezosensitive frigidimarina fidelis japonica 40 35 −50 −40 −30 −50 −40 −30 −50 −40 −30 −50 −40 −30 ← Increasing acidic pI bias (%) Supplementary Figure 4. Isoelectric point (pI) bias of proteins within each strain. A; Isoelectric point (pI) bias of proteins plotted as a function of % GC content within the genomes of members of the Colwellia, Psychromonas, and Shewanella. B; pI bias of members of the Shewanella plotted as a function of % GC when taking into account within-genus position broadly based on a phylogenetic tree generated by Alex & Antunes, 2019. Strains are organized by general clade grouping, labeled here as clades 1-4. A 120 80 Piezophile Piezosensitive 40 Pressure optimum (MPa) Pressure optimum 0 −40 −30 −20 −10 ← Increasing acidic pI bias (%) B 9000 6000 Piezophile Piezosensitive 3000 Depth of collection (m) 0 −40 −30 −20 −10 ← Increasing acidic pI bias (%) C 30 20 Piezophile Piezosensitive 10 Optimum Temperature (°C) Temperature Optimum 0 −40 −30 −20 −10 ← Increasing acidic pI bias (%) Supplementary Figure 5. Isoelectric point bias of members of the Colwellia as a function of optimum pressure of growth (A), depth of collection (B), or optimum temperature (C). Supplementary Figure 6. Growth of strains of Colwellia (piezophile, n=3; piezosensitive, n-=3) measured as optical density (OD600) as a function of copper (II) chloride dihydrate concentrations at 4°C and optimum pressure. The OD values are plotted as a percentage of the OD measured under optimal conditions, when no copper (II) chloride is added. A 500000 1000000 1500000 2000000 2500000 3000000 3500000 4000000 C. MT41 500000 1000000 1500000 2000000 2500000 3000000 3500000 4000000 C. MT2012.fas 500000 1000000 1500000 2000000 2500000 3000000 3500000 4000000 C. TT2012.fas 500000 1000000 1500000 2000000 2500000 3000000 3500000 4000000 4500000 5000000 C. piezophila ATCC BAA-637.fas 500000 1000000 1500000 2000000 2500000 3000000 3500000 4000000 4500000 5000000 5500000 C. psychrerythraea GAB14E.fas 500000 1000000 1500000 2000000 2500000 3000000 3500000 4000000 4500000 5000000 C. psychrerythraea ND2E.fas 500000 1000000 1500000 2000000 2500000 3000000 3500000 4000000 4500000 5000000 C. psychrerythraea 34H.fasta B Toxin/Antitoxin system Transposase Phage−related Genomic Island Piezo−enriched genes 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 Genome position (Mbp) Supplementary Figure 7. A; Mauve alignment of strains of Colwellia. C. marinimaniae MTCD1 is labeled as ‘C. MT2012’. B; Locations of specific genomic elements identified within Colwellia marinimaniae MT41. .
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